Technical Field
The present disclosure relates to the field of reagent delivery, and more particularly, to devices and methods delivering reagents in an automated and sequential manner.
Description of the Related Art
Various chemical, biological, or other applications can benefit from automated fluidic or reagent delivery. Examples include, and are not limited to RNA, DNA, protein extraction and purification from biological samples, hybridization of nucleic acid to microarrays, immunological assays, protein and western blots processing, or more generally separation of a chemical which requires incubation of a set of known reagent with separation media.
These processes are typically performed in well-equipped laboratories. They generally involve delivery of various reagents onto a target chamber which often times is the core or contains the active component for that process. For example, extraction of nucleic acid includes using an active component, which is the matrix inside a “spin column.” In microarray based hybridization the active component is an array of capture probes. These reagents can play many roles such as buffering or changing the PH of the core chamber contents or changing its salt concentration. In addition, or instead, they can act as washes, or be the solution in which the target of interest is dissolved. It is desirable these reagents be prepared, procured, mixed, and/or stored in sanitary environments and conditions, and delivered in accurate amounts to the process core in a predefined sequence and/or timing.
One such process example, SPE or solid phase extraction, is a separation process by which compounds, dissolved or suspended in fluid mixture, are separated from other compounds in the mixture according to their physical and chemical properties. Analytical laboratories use solid phase extraction to concentrate and/or purify samples for analysis.
For example, solid phase extraction can be used to isolate analytes of interest from a wide variety of sources, including, but not limited to, urine, blood, water, beverages, soil, and animal tissue. SPE uses the affinity of solutes dissolved or suspended in a liquid (known as the mobile phase) for a solid through which the sample is passed (known as the stationary phase) to separate a mixture into desired and undesired components. The result is that either the desired analytes of interest or undesired impurities in the sample are retained on the stationary phase. The portion that passes through the stationary phase is collected, transferred, or discarded, depending on whether it contains the desired analytes or undesired impurities. If the portion retained on the stationary phase includes the desired analytes, they can then be removed from the stationary phase for collection in an additional step, in which the stationary phase is rinsed with an appropriate eluent.
Existing devices and methods used to facilitate reagent delivery for various steps of aforementioned or other existing processes are generally time consuming, inefficient, and prone to operator error and environmental contaminants, and have cost drawbacks. For example, the classic manual pipetting of reagents is time-consuming, resource dependent, and expensive. Manual pipetting requires multiple manually performed steps of dispensing solutions such as suspensions and washing buffers, consuming scientist labor and requiring numerous tools and consumables such as multiple pipettes, pipette tips and vials. Other manually operated devices that attempt to improve on manual pipetting, continue to consume scientist labor and time, and are limited in application.
Two significant drawbacks of manual operation is operator error and environmental contaminants. Automatic pipetting similarly requires exceedingly expensive equipment. Certain existing automated or semi-automated devices are either intended for solely microfluidic applications or are otherwise extremely complicated and expensive to manufacture, purchase, and operate. Some automated devices require complicated internal mechanisms and/or requirements, which make it extremely difficult to load reagents.
An additional drawback of some existing devices includes the lack of capability to assemble, organize, or arrange reagents separate from the devices, and requiring the devices to serve as the reagent container.
By way of a particular example, the following is an existing RNA extraction procedure based on existing Qiagen™ RNeasy Mini spin columns™.
The RNeasy™ kit provides protocols and reagents for purification of total RNA from animal cells, animal tissues, plant cells and tissues, filamentous fungi and yeast, and for cleanup of RNA from crude RNA preps and enzymatic reactions (e.g., DNase digestion, proteinase digestion, RNA ligation, and labeling reaction).
The RNeasy™ Mini Kit can also be used to purify total RNA from bacteria. The purified RNA is suitable for downstream applications such as:                RT-PCR and real-time RT-PCR        Differential display        cDNA synthesis        Northern, southern, and western, dot, and slot blot analyses        Primer extension        Poly A+RNA selection        RNase/S1 nuclease protection        Microarrays.        
Summarized protocol:
1) Sample preparation:
Disrupt the sample according to the appropriate protocol in order to release RNA into solution. Most often, the solution is RLT lysis buffer. Add one volume of 70% ethanol to the lysis buffer and mix.
22) Binding.
Add up to 700 μl of solution (RLT+EtOH+sample) to the Qiagen™ spin column, close the lid, and centrifuge at ≥8000 rcf for 15 seconds.
3) Washes:
Remove the column from the collection tube, empty the flow-through, and return the column back to the collection tube. Add 700 μl RW1 wash buffer to the column, close the lid, and centrifuge at ≥8000 rcf for 15 seconds. Remove the column from the collection tube, empty the flow-through, and return the column back to the collection tube. Add 500 μl RPE (1:4 RPE concentrate to 100% EtOH, premixed) to the column, close the lid, centrifuge at ≥8000 rcf for 15 seconds. Remove the column from the collection tube, empty the flow-through, and return the column back to the collection tube. Repeat the RPE wash two more times. Dry the column by removing the column from the collection tube, wiping residual solution off of the column, putting the column into a clean collection tube, and centrifuging at ≥8000 rcf for 1 minute.
4) Elution:
Place the column into a new collection tube, add 30-50 μl of elution water, close the lid, and centrifuge at ≥8000 rcf for 15 seconds. The eluent will contain the RNA and is ready for downstream applications.
As demonstrated above, this is one example, which requires multiple steps, demanding scientist time and expensive components.